Histological sections prepared from cortical parts of 25 bovine ovaries were used to study initiation of follicle growth in vivo. Small follicles were measured and characterized. Initiation of follicle growth consisted of two distinct consecutive phases. The first phase was characterized by transformation of granulosa cells from a flattened to a cuboidal shape and by their proliferation. In the second phase an increase in the number of granulosa cells was accompanied by a rapid increase in the size of the oocyte. Oocytes commenced growth when there were at least 40 granulosa cells in the largest cross-section (fourth generation of follicle cells). The oocyte diameter increased from 29.74 ± 0.30 μm (mean ± sem) in primordial follicles to 92.90 ± 4.50 μm in small antral follicles. The zona pellucida first appeared as an island of periodic acid–Schiff positive material in small preantral follicles, but formed a complete ring around the oocyte when the late preantral stage was reached. Organ culture of ovarian cortical explants was used to study initiation of follicle growth in vitro. Within 2 days of culture most of the primordial follicles entered the growth phase: granulosa cells changed from a flattened to a cuboidal shape and entered S-phase as demonstrated by autoradiography after [³H]thymidine incorporation. On day 2, 48.6% of follicles were labelled compared with 3% on day 0. Follicle growth started in the absence of gonadotrophins, in the serum-free medium, confirming the notion that gonadotrophins are not essential for this process. The culture system used here will be helpful in the study of the involvement of putative factor(s) in the initiation of follicle growth in large domestic animals.

Summary. Booroola–Awassi ewe lambs were heterozygous (F +) for a major gene F, influencing their ovulation rate, while Awassi lambs were non-carriers (++). Basal plasma FSH concentration (mean ± s.e.m.) in Booroola–Awassi ewe lambs at 4 weeks of age was significantly higher than in Awassi lambs of the same age (5·06 ± 0·60 and 2·04 ± 0·32 ng/ml respectively; P < 0·001). After GnRH administration, FSH increased from 3·89 ± 1·10 to 10·58 ± 1·30 ng/ml in Booroola–Awassi (N = 6) and from 1·87 ± 0·29 to 4·64 ± 0·33 ng/ml in Awassi (N = 6) ewe lambs (P < 0·05). Ovariectomy caused an increase in plasma FSH in Booroola–Awassi (N = 4) and Awassi (N = 4) ewe lambs. At 1 week after ovariectomy plasma FSH increased from 5·96 ± 1·02 to 7·06 ± 1·05 ng/ml in F+ and from 1·67 ± 1·06 to 5·21 ± 0·66 ng/ml in ++ ewe lambs, suggesting a stronger negative feed-back effect exerted by the ovaries of Awassi lambs. At 15 weeks after ovariectomy FSH values were similar in Booroola-Awassi (18·28 ± 1·96 ng/ml) and Awassi (16·07 ± 0·70 ng/ml) lambs. Although the overall pattern of pituitary response to ovariectomy was similar in the F + and ++ ewe lambs, Booroola–Awassi lambs had small ovaries (132·5 ± 24·9 mg) and follicular development did not proceed beyond the preantral stage in 3/4 animals, and Awassi lambs had large ovaries (600·0 ± 233·9 mg) (P < 0·05) with many preantral and antral follicles.

The present results suggest that (1) the elevated plasma FSH concentration between 3 and 6 weeks of age in Booroola–Awassi compared to Awassi ewe lambs might be due to (a) greater responsiveness of the F + pituitary to GnRH and (b) reduced ovarian suppression of FSH release and (2) the ovaries of Booroola–Awassi lambs, although lacking antral follicles, secrete a factor that controls FSH values. We suggest that differences between the two genotypes can be attributed to the F gene or to other minor genes that came from the Booroola–Merino sires used to create the Booroola–Awassi progeny.

Keywords: sheep; pituitary; ovary; genotype

The onset of gene expression for three proteins that play pivotal roles in theca interna function, namely the LH receptor (LH-R), cytochrome P450 17α-hydroxylase (17αOH) and the steroidogenic acute regulatory protein (StAR), was determined. Ovaries were obtained on day 9 of the oestrus cycle from mature synchronized dairy cows (n = 5) and gene expression in preantral and antral follicles up to 4 mm in diameter was evaluated by in situ hybridization. LH-R and 17αOH mRNAs were observed first, in the theca interna of large preantral follicles (type 4), concurrent with its morphological differentiation. StAR mRNA appeared later during follicular growth, in follicles >1 mm in diameter (type 6). LH-R and 17αOH mRNAs were found exclusively in the thecal cells, whereas StAR mRNA appeared in thecal cells, granulosa cells of late atretic follicles and oocytes. In early atresia, thecal cells expressed all three mRNAs, and their expression decreased gradually as atresia progressed. Atresia in granulosa cells was characterized by massive apoptosis of periantral, but not peribasal cells, that differentiated into luteal-like cells expressing StAR.

In summary, our study suggests that in spite of the presence of 17αOH, a key enzyme in steroidogenesis, the ability to produce steroids by bovine follicles smaller than 1 mm in diameter must be very limited due to the absence of StAR protein. During the early stages of atresia, thecal cells remain morphologically and functionally healthy, and continue to express all three studied mRNAs.

The aim of this study was to characterize the immediate effects of heat stress on plasma FSH and inhibin concentrations, and its involvement in follicular dynamics during a complete oestrous cycle, and to examine a possible delayed effect of heat stress on follicular development. Holstein dairy cows were oestrous synchronized and randomly assigned to either cooled (n = 7) or heat-stressed (n = 6) treatment groups. During a complete oestrous cycle, control cows, which were cooled, maintained normothermia, whereas heat-stressed cows, which were exposed to direct solar radiation, developed hyperthermia. At the end of this oestrous cycle (treated cycle), both groups were cooled and maintained normothermia for the first 10 days of the subsequent oestrous cycle. Throughout this period, follicular development was examined by ultrasonography, and plasma samples were collected. During the second follicular wave of the treated oestrous cycle, a significantly larger cohort of medium sized follicles (6-9 mm) was found in heat-stressed cows than in cooled cows (P < 0.05). The enhanced growth of follicles in this wave in heat-stressed cows was associated with a higher plasma FSH increase which lasted 4 more days (days 8-13 of the oestrous cycle; P < 0.05), and coincided with a decrease in the plasma concentration of immunoreactive inhibin (days 5-18 of the oestrous cycle; P < 0.05). During the follicular phase (days 17-20 of the treated cycle), heat-stressed cows showed an increase in the number of large follicles (>/= 10 mm), and the preovulatory plasma FSH surge was significantly higher in heat-stressed cows than in cooled cows (P < 0.01). The effect of heat stress was also observed during the first follicular wave of the subsequent cycle: the postovulatory plasma FSH concentration was higher (P < 0.01), but fewer medium follicles developed, and the first follicular wave decreased at a slower rate in previously heat-stressed cows than in cooled cows (0.40 and 0.71 follicles per day, respectively). This study shows both immediate and delayed effects of heat stress on follicular dynamics, which were associated with high FSH and low inhibin concentrations in plasma. These alterations may have physiological significance that could be associated with low fertility of cattle during the summer and autumn.

During the autumn, the conception rate of dairy cattle in warm countries is low although ambient temperatures have decreased and cows are no longer exposed to summer thermal stress, indicating that there may be a delayed effect of heat stress on cattle fertility. Two experiments were conducted to examine possible delayed effects of heat stress on follicular characteristics and steroid production at two distinct stages of follicular growth: medium-sized and preovulatory follicles, 20 and 26 days after heat exposure, respectively. Lactating cows were subjected to heat stress for 12 h a day in an environmental chamber, during days 2-6 of a synchronized oestrous cycle. In Expt 1, ovaries were collected on day 3 of the subsequent cycle, before selection of the dominant follicle, and medium-sized follicles were classified as atretic or healthy. In Expt 2, on day 7 of the subsequent cycle, PGF(2a) was administered and preovulatory follicles were collected 40 h later. In both experiments, follicular fluid was aspirated, granulosa and thecal cells were incubated, and steroid production was determined. In healthy medium-sized follicles (Expt 1), oestradiol production by granulosa cells and androstenedione production by thecal cells were lower (P < 0.05) and the concentration of progesterone in the follicular fluid was higher in cows that had been previously heat-stressed than in control cows (P < 0.05). In preovulatory follicles (Expt 2), the viability of granulosa cells was lower (P < 0.05) and the concentration of androstenedione in the follicular fluid and its production by thecal cells were lower (P < 0.05) in cows that had been previously heat-stressed than in control cows. In both experiments, the oestradiol concentrations in the follicular fluids were not altered by heat stress. These results demonstrate a delayed effect of heat stress on steroid production and follicular characteristics in both medium-sized and preovulatory follicles; this effect could be related to the low fertility of cattle in the autumn.

The fertility of dairy cows decreases during the summer and remains low during the cooler autumn although the animals are no longer under heat stress. The aim of this study was to characterize a delayed effect of summer heat stress on oocyte quality in the autumn and to improve oocyte quality by enhanced removal of follicles damaged during the previous summer. Lactating cows (n = 16) were subjected to heat stress during the summer. In autumn, ovarian follicles (3-7 mm in diameter) were aspirated by an ultrasound-guided procedure during four consecutive oestrous cycles. Follicles were aspirated from control cows on day 4 and from treated cows on days 4, 7, 11 and 15 of each oestrous cycle. All cows received PGF(2alpha) and GnRH injections on days 19 and 21, respectively, and maintained cyclicity, as indicated by plasma progesterone concentrations. On day 4 of each cycle, the oocytes recovered were examined morphologically, matured and activated in vitro, and cultured for 8 days. In cycle 1 (early October) both groups showed low percentages of grade 1 oocytes, cleavage, four- and eight-cell embryos, morulae and parthenogenetic blastocysts. Subsequently, the number of grade 1 oocytes increased earlier (cycle 2) in treated than in control cows (cycle 3; P < 0.05). The cleavage rate in the control group remained relatively low throughout (32-58%), whereas in the treated group it increased from 40% (cycle 1) to 75% (cycles 3 and 4; P < 0.05). The number at each stage of embryo development increased slightly but remained low throughout in the control group, whereas in the treated group significant (P < 0.05) increases of all stages were observed in cycles 3 and 4. The results show a delayed effect of summer heat stress on oocyte quality and embryo development in the autumn. Enhanced removal of the impaired cohort of follicles led to earlier emergence of healthy follicles and high quality oocytes in the autumn.

The aims of this study were to examine the effects of the Booroola Fec ^B gene on ovarian development and reproductive hormones (FSH, LH and inhibin) at days 90, 100, 120 and 135 of gestation (term = 147 days). The effects of litter size were eliminated by transferring equal numbers of homozygous BB and control (++) embryos to recipient ewes. The ovary, but not the body, pituitary, adrenal, kidney or thymus, was heavier (P < 0.05) in BB compared with ++ fetuses at day 90 but not thereafter. In the ovary, gene-specific differences were observed in the total number of germ cells present at days 90 (P< 0.01) and 135 (P < 0.05) with the same tendency being noted at day 100 (P < 0.07); at all of these ages the mean numbers of germ cells in the BB genotype exceeded those in ++ animals. Gene-specific differences were observed in the numbers of oogonia and isolated oocytes at day 90 (i.e. BB > ++), in the number of primordial follicles at days 100 (BB > ++) and 135 (BB > ++), and also in the number of primary or secondary follicles (++ > BB) at day 135. At each gestational age examined no differences were noted with respect to the plasma concentrations of FSH, LH or inhibin between the BB and ++ fetuses. However, the highest mean plasma concentrations of FSH and LH occurred at days 90 and 100 of gestation, which coincided with the first developing primary follicles. Collectively, the results from this and previous studies show that the different effects of the Fec ^B gene in germ cell development in early gestation continue throughout fetal development independently of litter size. Moreover, during the growth of the first primary and secondary follicles at days 100 and 120, respectively, there are no differences in the plasma concentrations of FSH, LH and inhibin with respect to Booroola genotype.